Method of elevating yield of oligosacchatides containing alpha -galactosyl and anti-candida compositions

ABSTRACT

A novel method of very efficiently elevating the yield of oligosacchrides containing α-galactosyl, compared with the conventional methods, which comprises treating galactose or a galactose-containing material with a specific α-galactosidase and thus performing a dehydrocondensation reaction at a high substrate concentration.  
     Anti-candida compositions originating in foods, having a high safety and excellent anti-candida effect and not being restricted in the supply, which contain as the active ingredient oligosacchrides obtained by treating galactose or a galactose-containing material with an α-galactosidase and thus performing a dehydrocondensation.

FIELD OF THE INVENTION

[0001] The present invention relates to a method of elevating the yieldof an oligosacchride containing α-galactosyl by treating galactose or agalactose-containing material with a specific microorganism-derivedgalactosidase and an anti-candida composition comprising as an activeingredient an oligosacchride containing α-galactosyl other thanraffinose.

BACKGROUND OF THE INVENTION

[0002] Recently, various saccharide-related enzymes which synthesizescertain oligosaccharides were screened for on the basis ofmicroorganisms, and the advancement in a technology for utilizing such amicroorganism-derived enzyme and in a technology for purifying aresultant synthetic oligosaccharide extensively enables a large scaleproduction of the oligosaccharide at a low cost. Accordingly, anoligosaccharide became utilizable even in a field familiar to us such asa food industry. Those known typically are a coupling sugar,fractooligosaccharide, β-galactooligosaccharide, soybeanoligosaccharide, isomaltooligosaccharide, palatinose, lactosucroseoligosaccharide and the like.

[0003] Any of these oligosaccharide was reported to have a bifidusmicroorganism growth activity or a cariostatic or anti-cariogenicproperty as well as a mineral absorption-promoting effect, and isutilized as a specified health-care food or a starting material thereof.

[0004] It is also reported recently that an oligosacchride containingα-galactosyl such as melibiose, manninotriose, raffinose and stachyosehas a potent proliferating effect on a bifidus microorganism, has afavorable property as a food, and have a carcinostatic effect or naturalkiller cell-activating effect (Shigeki Motoi et al, Japanese CancerAssociation (meeting report), 40, 132 (1981)), and such anoligosaccharide is considered to be extremely useful and attractive as abeverage, food product, pharmaceutical and a starting material thereof.

[0005] A known source of raffinose is a beet or soybean oligosaccharide,while that of stachyose is a soybean oligosaccharide. However, a soybeanoligosaccharide is not found in abundance, and can not be supplied in alarge amount. Besides, it is costly. Raffinose contained in a beet isalso disadvantageous since it is available only during a period ofOctober to March, its annual supply is only several 100 tons and itscurrent price is so high. Melibiose and manninotriose are present insmall amounts in a soybean oligosaccharide, and have the structuresformed by deleting a fructose moiety from raffinose and stachyose,respectively. While they are synthesized artificially by means of thedecomposition of raffinose and stachyose, they are very expensive sincethe starting raffinose and stachyose are also expensive.

[0006] On the other hand, an enzyme reaction employing anα-galactosidase may also be employed for synthesizing an oligosacchridecontaining α-galactosyl. Such an enzyme reaction is classified into asaccharide transfer reaction and a dehydrocondensation reaction.

[0007] The saccharide transfer reaction is not suitable as an industrialmethod since it requires a synthesis substrate such as p-nitrophenylα-galactoside as an α-galactosyl group donor (JP-A-10-201472) and alsorequires as a substrate an expensive compound containing α-galactosylsuch as raffinose.

[0008] When utilizing the dehydrocondensation reaction, it is possibleto conduct a reaction at a high substrate concentration, and a freegalactose employed as a substrate may be one obtained by a hydrolysis ofa less expensive saccharide such as lactose, whereby accomplishing aneconomically advantageous reaction.

[0009] An enzyme which can be employed in this dehydrocondensationreaction is proposed to be an α-galactosidase produced by amicroorganism such as Pycnoporus cinnabarinus, Streptococcus bovis,Diplococcus pneumoniae, Mortierella vinacea, Pseudomonas fluorescensstrain H-601 (deposition No: FERM P-11027) and Candida guilliermondiistrain H-404 (deposition No: FERM P-11026) as well as a plant such asVicia sativa and Green coffee bean (Japanese Patent No.3028258).

[0010] However, these enzymes are not applied to an industrial scaleproduction since they exhibit low reaction rates and low reaction yieldsat high substrate concentrations. Thus, the α-galactosidase produced byCandida guilliermondii strain H-404 which has been believed to exhibitthe highest reaction rate and the highest reaction yield requires aperiod, for example, as long as about 90 hours until its reaction isplateaued when being treated with 60% (w/v) galactose under theconditions of 35 U^(M)/g-galactose and 50° C., but only gives an yieldof oligosacchrides containing a-galactosyl as low as about 25% (AppliedScience of Saccharide, 44, 69-75, 1997).

[0011] As described above, an oligosacchride containing α-galactosylhaving useful characteristics as a food material is not supplied in alarge amount or at a low cost and desired to be produced on anindustrial scale.

[0012] The invention is intended to solve the problem that aconventional method poses a low reaction rate and a low reaction yieldin a dehydrocondensation reaction at a high substrate concentration andis less practical, and its objective is to provide a method forproducing an oligosacchride containing α-galactosyl far more efficientlythan any conventional method by using a certain α-galactosidase.

[0013] On the other hand, a Candida microorganism is one of the yeastswhich are persistent in the intestine of a human, and is anopportunistic pathogen inducing a serious systemic infections when theresistance is reduced due to an infectious disease or other diseases. Anintestinal Candida microorganism exerts its effect on an immune systemvia the cell itself or a Candida toxin produced by the microorganism,and was reported to serve as an atopic dermatitis exacerbation factor(Michio Matsuda et al., allergy clinics, 56, 768-772 (1991)) or to beinvolved in the allergic diseases other than the atopic dermatitis, suchas bronchial asthma and rhinitis. Otherwise, it induces, whenproliferating abnormally in an intestine, a health problem such as achronic fatigue called yeast connection, headache, uneasiness,unsettledness, depression and the like.

[0014] As a method for treating such a Candida-induced mycosis or atopicdermatitis, an antifungal therapy is considered to be effective, and itwas reported recently that a trisaccharide raffinose occurring in a beetplant has not only in vitro but also in vivo inhibitory and eradicativeeffects on a Candida microorganism (JP-A-11-240837) and actually exertedan effect on an atopic dermatitis (Taizo Nagura et al, Food Industries,2.28, 29, 1999). An oral administration of raffinose does not cause sideeffects observed with an antifungal agent such as a transientexacerbation of rash or digestive organ signs, and is regarded as asafer therapy against an atopic dermatitis.

[0015] Nevertheless, a further potent and further safer therapy has beendemanded since the anti-candida effect of raffinose is lower than thatof an antifungal agent. Also since a Candida microorganism has variousadverse effects on a human health as described above, a food producthaving a more potent anti-candida effect has been desired from aviewpoint of a diet-based health control trend in response to the demandof reducing the medical expense. Another problem associated withraffinose is its limited supply and expensiveness as described above.

[0016] On the other hand, the physiological functions of anoligosacchride containing α-galactosyl other than raffinose were alsoreported intensively, including a neutralizing effect ofα-1,3-galactobiose (α-1,3 Gal2) on a toxin produced by Clostridiumdifficile which is a causative bacteria of a colitis (L. D. Heerze etal., J.Infect.Dis, 169, 1291-1296 (1994)) and an inhibitory andtherapeutic effect of α-1,4 galactobiose (α-1,4 Gal2) on a pathogenicE.coli 0157 (C. A. Lingwood, Adv. Lipid Res., 25, 189-211 (1993)).

[0017] Nevertheless, no reports suggested any anti-candida effect of anoligosacchride containing α-galactosyl other than raffinose.

[0018] Also since α-galactosyl group is present only in the form of anα-galactobiose (α-Gal2) in a living body and the α-1,6-galactosylglucose structure possessed by raffinose does not exist while themechanisms of the anti-candida effect of raffinose are considered to bebased on the intestinal flora-improving effect, the inhibitory effect onthe adhesion or settlement of a Candida microorganism on a digestivetract and the immune activation effect, we assumed that α-Gal2 plays animportant role in the in vivo effect of raffinose and focused on anoligosacchride containing α-galactosyl other than raffinose, wherebyaccomplishing the invention.

[0019] Accordingly, an objective of the invention is to provide ananti-candida composition which is derived from a food, highly safe,excellent in anti-candida effect, and can be supplied limitlessly.

SUMMARY OF THE INVENTION

[0020] The invention relates to a method of elevating the yield of anoligosacchride containing α-galactosyl comprising treating galactose ora galactose-containing material with an α-galactosidase derived from aspecific microorganism, and such an microorganism which produces anα-galactosidase employed in the invention may, for example, be a fungussuch as Aspergillus, Penicillium and Trichoderma, yeast such asSaccharomyces and a bacterium such as Bacillus, with an α-galactosidasederived from a microorganism of Aspergillus being preferred.

[0021] Among the microorganisms listed above, those employed preferablyare Aspergillus niger, Aspergillus oryzae and Aspergillus pulverulentusamong the fungi of Aspergillus, Penicillium citrinum and Penicilliummulticolor among the fungi of Penicillium and Trichoderma viride amongthe fungi of Trichoderma, with Aspergillus niger being preferredespecially.

[0022]Saccharomyces cerevisiae is preferred among the yeasts ofSaccharomyces, and Bacillus megaterium is preferred among the bacteriaof Bacillus.

[0023] Moreover, it was also discovered that a fungus we isolated from asoil produces a novel α-galactosidase which is utilizable in theinvention.

[0024] This strain has the following mycological characteristics.

[0025] Conidial top: Spherical to radial.

[0026] Conidium (optical microscope): Spherical to semi-spherical,smooth to slightly rough surface.

[0027] Conidium (electron microscope): Rough surface (protrusion),diameter (about 3.5 to 4.0 μm).

[0028] Exudate: Slight (transparent to tan).

[0029] Odor: Almost none

[0030] Sterigmata: 2-Stage.

[0031] Colony formation rate (malt extract agar medium, incubated at 25°C.): >85 mm (7-day incubation), >85 mm (12-day incubation).

[0032] Colony growth rate (Czapek's yeast agar medium, incubated at 25°C.): 60 to 67 mm (7-day incubation), 65 to 73 mm (12-day incubation).

[0033] Colony color (malt extract agar medium): Black (front), colorless(back)

[0034] Colony color (Czapek's yeast agar medium): Black to grayish black(front), cream to grayish yellow (back)

[0035] This strain is classified as Aspergillus niger ver.niger based onthe conidial shape, sterigmata and colony color. We designated thisstrain as Aspergillus niger strain APC-9319. This strain was depositedunder the deposition number of FERM BP-7680 to International PatentOrganism Depositary of National Institute of Advanced Industrial Scienceand Technology (IPOD) (transfer from domestic deposition tointernational deposition, Original deposition date: Aug. 29, 2000,Domestic deposition number: FERM P-18003).

[0036] This novel α-galactosidase has the following physicochemicalcharacteristics:

[1] Effect

[0037] It catalyzes a reaction for hydrolyzing an α-galactoside bond toliberate D-galactose:

Gal1α-OR+H₂O→Gal-OH+R—OH

[0038] wherein Gal1α-OR is a saccharide containing α-galactosyl, Gal-OHis a free galactose, and R—OH is a compound containing hydroxyl such asvarious saccharides, alcohols and phenols);

[2] Substrate Specificity

[0039] It acts on a saccharide having an α-galactosyl group at itsnon-reducing terminal such as melibiose, raffinose and stachyose as wellas p-nitrophenyl α-galactoside, with the relative rate at whichmelibiose is decomposed being about 9 based on the decomposition rateusing p-nitrophenyl α-galactoside as a substrate being regarded as 100;

[3] Optimum pH and pH Stability

[0040] Its optimum pH is 2.5 to 6.0, and it is stable within the rangefrom pH 3.5 to 8.0 when allowed to stand for 1 hour at 40° C.;

[4] Optimum Temperature and Heat Stability

[0041] Its optimum temperature at pH 4.5 (acetate buffer) is 60° C., andit is stable at a temperature not higher than 60° C. when allowed tostand at pH 4.5 (acetate buffer) for 15 minutes;

[5] Molecular Weight and Isoelectric Point

[0042] Its molecular weight measured by a gel filtration method using aYMC-Pack Diol-200 column (YMC) is 217,000 and its molecular weightmeasured by an SDS-PAGE is 117,000, while its isoelectric point measuredby an isoelectric focusing is 4.2.

[0043] When producing an α-galactosidase using any of thesemicroorganisms, a solid culture or liquid culture is usually employed.

[0044] A medium for a solid culture is a wheat bran itself or a wheatbran supplemented with various additives including organic and inorganicnitrogen compounds, such as KINAKO powder, soybean powder, ammoniumsalt, nitrate, urea, glutamic acid, aspartic acid, polypeptone, cornsteep liquor, meat extract, yeast extract, protein hydrolysate and thelike. In addition, suitable inorganic salts may also be added.

[0045] A medium for a liquid culture may for example be a syntheticmedium or natural medium containing carbon sources, nitrogen sources,inorganic salts and essential nutritions which are required for afavorable growth of a relevant microorganism and a satisfactoryproduction of an enzyme. For example, a carbon source may be acarbohydrate such as a starch or constituent thereof, roasted dextrin,processed starch, starch derivative, physically processed starch and astarch or a galactose-containing material. Typically, a soluble starch,corn starch, potato starch, sweet potato starch, dextrin, amylopectin,amylose, galactose, lactose, raffinose, and the like, any of which canbe employed alone or in combination with each other.

[0046] A nitrogen source may for example be an organic nitrogen sourcematerial such as a polypeptone, casein, meat extract, yeast extract,corn steep liquor or soybean extract or soybean lee extract, aninorganic salt nitrogen compound such as ammonium sulfate and ammoniumphosphate, and an amino acid such as glutamic acid, any of which can beemployed alone or in combination with each other.

[0047] An inorganic salt may for example be a phosphate such asmonopotassium phosphate and dipotassium phosphate, a magnesium salt suchas magnesium sulfate, a calcium salt such as calcium chloride and asodium salt such as sodium carbonate, any of which can be employed aloneor in combination with each other.

[0048] A solid culture is conducted without shaking at a pH of theculture medium within the range from 3 to 7, preferably 4 to 7, to whichan inventive microorganism is inoculated and cultured at 10 to 40° C.,preferably 20 to 37° C. for 1 to 10 days. After incubation, the culturemedium extract was subjected to an ethanol precipitation and the like toobtain α-galactosidase as a crude enzyme precipitate.

[0049] A liquid culture is conducted with shaking or with aerating andstirring under an aerobic condition, at a pH of the culture mediumwithin the range from 4 to 10, preferably 5 to 8 at a temperature of 10to 40° C., preferably 25 to 37° C., for a period of 24 to 96 hours.After incubation, the cells were removed by centrifugation or othersuitable solid-liquid separation means to obtain a culture supernatant.Alternatively, the cells were treated physically or enzymatically toobtain an intracellular extract.

[0050] Then, such a crude enzyme solution is subjected to a suitablecombination of an ammonium sulfate salting out, gel filtration,hydrophobic interaction chromatography and the like to obtain a highlypure α-galactosidase.

[0051] An enzyme used for producing an oligosacchride containingα-galactosyl may not only be the enzyme preparations obtained by theabove-described method but also an extract of a solid culture and aculture supernatant or intracellular extract of a liquid culture whichmay be employed as enzyme formulations as they are. If necessary, anenzyme obtained by a known purification method may also be employed. Itis also possible to use a cell itself as an enzyme formulation.Otherwise, an α-galactosidase present as a contaminant in a commerciallyavailable enzyme formulation, such as a cellulase or proteaseformulation may also be employed, and in such a case the enzymeformulation may be used as it is or subjected to a known purificationprocess to isolate the intended α-galactosidase.

[0052] Examples of the commercially available enzyme formulationproducts employed for producing an oligosacchride containingα-galactosyl are listed below: Aspergillus niger-derived α-GalactosidaseS-DS (6,500 U/g), lipase A “AMANO” 6 (231 U/g), hemicellulase “AMANO”90G (97 U/g) or cellulase A “AMANO” 3 (26 U/g), Aspergillusoryzae-derived “UMAMIZYME” (163 U/g), protease M “AMANO” (3 U/g),Aspergillus pulverulentus-derived pectidase G “AMANO” (131 U/g),Penicillium citrinum-derived protease B “AMANO” (235 U/g) or nuclease“AMANO” (388 U/g), Trichoderma viride-derived cellulase “AMANO” 4 (6U/g) and the like.

[0053] Among those listed above, Aspergillus niger-derivedα-Galactosidase S-DS (6,500 U/g), Penicillium citrinum-derived proteaseB “AMANO” (235 U/g) and nuclease “AMANO” (388 U/g) are preferred.

[0054] All of the commercially available enzyme formulations describedabove are the products by AMANO ENZYME, and the number of each tradename represents the activity level of the α-galactosidase contained. Theactivity was measured by the method in Reference Example 1 describedbelow using an acetate buffer, pH6.0.

[0055] Such an enzyme or a cell which produces such an enzyme can beimmobilized and utilized repetitively in the reaction in a continuous orbatch process.

[0056] A starting material to be subjected to an α-galactosidasereaction may for example be galactose or a galactose-containingmaterial. Those exemplified typically are galactose, a mixture ofgalactose and glucose, a hydrolysate of a galactose-containing compoundsuch as lactose and the like, any of which can be employed alone or incombination with each other. The galactose is not only a commerciallyavailable galactose but also a galactose obtained by hydrolyzing anaturally occurring or synthetic oligosaccharide containing α-galactosylor β-galactosyl, glucoside or polysaccharide such as melibiose,manninotriose, raffinose, stachyose, planteose, verbascose, galactans,galactomannan, arabinogalactan, rhamnogalactan, galactolipid, ferulatedgalactose, galactopinitol, galactosylglycerol, galactinol, lactose,lactitol, lactulose, galactooligosaccharide and the like with an enzyme(β-galactanase, β-galactosidase, α-galactosidase and the like) or anacid.

[0057] The ratio of galactose and glucose in the mixture is not limitedparticularly, as far as galactose is contained.

[0058] Glucose, which is to be combined with galactose, is not only acommercially available glucose but also a glucose obtained byhydrolyzing a naturally occurring or synthetic oligosaccharidecontaining α-glucosyl or β-glucosyl, glucoside or polysaccharide such asstarch, maltooligosaccharide, isomaltoligosaccharide,nigerooligosaccharide, kojic oligosaccharide, cyclodextrin, trehalose,maltitol, cellulose, cellooligosaccharid, sophorooligosaccharide,laminaologosaccharide, gentiooligosaccharide and the like with an enzyme(amylase, glucoamylase, cellulase, α-glucosidase, β-glucosidase and thelike) or an acid. The compound which is to be combined with galactose isnot limited to glucose, and may be any other saccharide.

[0059] As a hydrolysate of a compound containing galactose, a materialobtained by hydrolyzing a naturally occurring or syntheticoligosaccharide containing α-galactosyl or β-galactosyl, glucoside orpolysaccharide such as melibiose, manninotriose, raffinose, stachyose,planteose, verbascose, galactans, galactomannan, arabinogalactan,rhamnogalactan, galactolipid, ferulated galactose, galactopinitol,galactosylglycerol, galactinol, lactose, lactitol, lactulose,galactooligosaccharide and the like with an enzyme (β-galactanase,β-galactosidase, α-galactosidase and the like) or an acid can beutilized as it is. Among those listed above, a lactose hydrolysateobtained by hydrolyzing a less expensive lactose with β-galactosidase oran acid is utilized preferably as it is. In addition, a lactosehydrolysate may be supplemented with galactose or glucose describedabove, or its ratio between galactose and glucose can be changed usingan ion exchange chromatography, an activated charcoal column and thelike.

[0060] While an α-galactosidase is a hydrolytic enzyme naturally, itcatalyzes a dehydrocondensation reaction, which is a reverse reaction ofthe hydrolysis, at an elevated level of galactose as a starting materialwhich serves as a substrate. Accordingly, when galactose at a high levelis treated with an α-galactosidase, oligosacchrides containingα-galactosyl which are compositions consisting of variousoligosaccharides such as disaccharides, trisaccharides andtetrasaccharides having a structure represented by α-(Gal)n(wherein n isusually an integer of 2 to 10, Gal is galactose) are formed. The sitewhere an α-galactosyl group is bound at the non-reducing terminal ofsuch an oligosacchride containing α-galactosyl is exemplified below.

[0061] [1] α-(Gal)n containing an α-1,6-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 2 to 10).

[0062] [2] α-(Gal)n containing an α-1,3-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 2 to 10).

[0063] [3] α-(Gal)n containing an α-1,2-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 2 to 10).

[0064] [4] α-(Gal)n containing an α-1,4-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 2 to 10).

[0065] The number of the bindings (n) is preferably 2 to 8, morepreferably 2 to 6.

[0066] When glucose is present in the reaction system, an oligosacchridecontaining α-galactosyl having a structure represented by α-(Gal)n-Glc(wherein n is usually an integer of 1 to 9, Glc is glucose) resultingfrom the binding between galactose and glucose is formed in addition toan oligosaccharide having a structure represented by α-(Gal)n. The sitewhere an α-galactosyl group is bound at the non-reducing terminal ofsuch an oligosacchride containing α-galactosyl is exemplified below.

[0067] [5] α-(Gal)n-Glc containing an α-1,6-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 1 to 9).

[0068] [6] α-(Gal)n-Glc containing an α-1,3-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 1 to 9).

[0069] [7] α-(Gal)n-Glc containing an α-1,2-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 1 to 9).

[0070] [8] α-(Gal)n-Glc containing an α-1,4-galactosyl group at thenon-reducing terminal (wherein n is usually an integer of 1 to 9).

[0071] The number of the bindings (n) is preferably 1 to 8, morepreferably 1 to 6.

[0072] In the procedure described above, the separation of anoligosaccharide from an enzyme reaction solution after completion of thereaction can be accomplished by a known separation means such as an ionexchange chromatography, activated charcoal column chromatography, gelfiltration column chromatography and the like.

[0073] Since the advancement of an enzyme reaction is accompanied with asaccharide transfer reaction due to the α-galactosidase-catalyzedre-decomposition of an oligosacchride ontaining α-galactosyl which wassynthesized previously by a dehydrocondensation reaction, the saccharidetransfer reaction also contributes to the synthesis of theoligosacchride containing α-galactosyl.

[0074] The site and the number of the binding of galactose in aresultant compound or the ratio of such a compound may vary depending onthe ratio between galactose and glucose in the starting material, originof the enzyme employed or reaction conditions.

[0075] While the condition under which an α-galactosidase is reacted mayvary depending on the enzyme employed, the reaction pH is 3.0 to 10.0,preferably 4.0 to 9.0.

[0076] The reaction temperature is preferably high in view of thesolubility and the reaction rate, and usually 20 to 90° C., preferably40 to 80° C. The reaction time may vary depending on the mount of anenzyme employed, and is usually 1 to 150 hours. Nevertheless, theinvention is not limited to the conditions or reaction modes describedabove.

[0077] When an oligosacchride ontaining α-galactosyl of the invention isproduced, a higher concentration of galactose as a starting material ismore preferred, and galactose or glucose may precipitate in the reactionsystem, or be present in a supersaturated condition, and used as aconcentration usually of 5 to 110% (w/v), preferably 40 to 110% (w/v).

[0078] For the purpose of an efficient production of an oligosacchridecontaining α-galactosyl by elevating galactose concentrationsubstantially, galactose is treated with an α-galactosidase to effect adehydrocondensation reaction as a preliminary reaction and thereafterthe reaction solution is concentrated under reduced pressure prior tothe dehydrocondensation reaction as a main reaction. This reaction heremay be conducted in a system separate from the preliminary reaction byisolating the concentrated solution, or may be conducted in thepreliminary reaction system without isolating the concentrated solution.

[0079] When the level of galactose is elevated at the stage of theinitial charging, galactose undergoes precipitation, which may lead to areduction in the yield of an oligosacchride containing α-galactosyl.Nevertheless, the inventive method in which the galactose level iselevated substantially to effect a dehydrocondensation reaction asdescribed above prevents any precipitation of galactose or anyinactivation of the enzyme due to the foaming upon concentration andpromotes the dehydrocondensation reaction markedly, thus allowing anoligosacchride containing α-galactosyl to be produced efficiently withina short period of time at a high yield. An enzyme employed in thisproduction method is preferably one having a higher catalytic activityof the dehydrocondensation reaction, such as those derived from variousmicroorganisms employed in an inventive yield elevating method amongwhich the α-galactosidase obtained from Aspergillus niger strainAPC-9319 (FERM BP-7680) is particularly preferred and can produce anoligosacchride containing α-galactosyl efficiently. The concentrationunder reduced pressure can be accomplished by any known means forconcentration under reduced pressure such as a rotary evaporator and avacuum crystallization container

[0080] The invention also relates to an anti-Candida compositioncomprising as an active ingredient an oligosacchride containingα-galactosyl other than raffinose which is synthesized by treatinggalactose or a galactose-containing material with an α-galactosidasefollowed by a dehydrocondensation reaction.

[0081] While the origin or type of a microorganism which produces anα-galactosidase employed for synthesizing an oligosacchride containingα-galactosyl of the invention is not limited particularly, and may beone derived from a plant such as a green coffee, any of variousα-galactosidases derived from the microorganisms employed in aninventive yield elevating method, especially an αgalactosidase derivedfrom Aspergillus niger strain APC-9319 (FERM BP-7680), serves to elevatethe yield of the oligosacchride containing α-galactosyl, wherebyenabling a stable and less expensive supply of a food-derived highlysafe anti-Candida composition. In addition, the synthesis may beaccomplished also by treating the galactose described above with anα-galactosidases to effect a dehydrocondensation reaction as apreliminary reaction followed by concentrating the reaction solutionunder reduced pressure prior to the dehydrocondensation reaction as amain reaction.

[0082] An oligosacchride containing α-galactosyl of the invention may bean oligosacccharide represented by α-(Gal)n (wherein n is usually aninteger of 2 to 10, Gal is galactose) consisting only of galactose or anoligosaccharide represented by α-(Gal)n-Glc (wherein n is usually aninteger of 1 to 9, Glc is glucose) consisting of other saccharide exceptfor raffinose, such as galactose and glucose.

[0083] An anti-Candida composition of the invention can be employed as asolid, semi-solid or liquid formed by combining an oligosacchridecontaining α-galactosyl other than raffinose with a customary inorganicor organic carrier or excipient. While an ordinary administration modeis an oral administration, a parenteral dosage form such as a dermalformulation can also be employed. An oral dosage form may be variousformulations such as powder, granule, tablet, capsule, syrup and thelike, and the formulation may contain pharmaceutically acceptable knownadditives such as an excipient, stability, binder, lubricant,disintegrant, suspending agent, solubilizer, flavor, sweetener and thelike.

[0084] The dose of an anti-Candida composition of the invention isusually 1 to 15 g per day, preferably 3 to 15 g per day as anoligosacchride containing α-galactosyl as an active ingredient whengiven orally to an adult. Since the oligosacchride containingα-galactosyl as an active ingredient is produced from a materialconsumed as a food by a method applied to a food product and has noproblem toxicologically and exhibited no acute toxicity in rats whengiven orally at 5000 mg per kg of body weight, its dose may be increasedor decreased as appropriate depending on the condition, age, bodyweight, dosage form, administration mode and the like.

[0085] An anti-Candida composition of the invention can be applied to amammal other than human such as cattle and horse as well as a laboratoryanimal such as rat and mouse, to which an oligosacchride containingα-galactosyl whose purity is lower may also be given.

BRIEF DESCRIPTION OF THE DRAWINGS

[0086]FIG. 1 shows the action pH and the optimum pH of theα-galactosidase obtained in EXAMPLE 1.

[0087]FIG. 2 shows the pH stability of the α-galactosidase obtained inEXAMPLE 1.

[0088]FIG. 3 shows the action temperature and the optimum temperature ofthe α-galactosidase obtained in EXAMPLE 1.

[0089]FIG. 4 shows the thermostability of the α-galactosidase obtainedin EXAMPLE 1.

[0090]FIG. 5 shows the results of the measurement of the molecularweight of the α-galactosidase obtained in EXAMPLE 1 measured by an HPLC.

[0091]FIG. 6 shows the calibration curve in the measurement of themolecular weight of the α-galactosidase obtained in EXAMPLE 1 by anHPLC.

[0092]FIG. 7 shows the results of the measurement of the molecularweight of the α-galactosidase obtained in EXAMPLE 1 measured by aSDS-PAGE.

[0093]FIG. 8 shows the isoelectric point, according to the isoelectricfocusing, of the α-galactosidase obtained in EXAMPLE 1.

[0094]FIG. 9 shows the change with time in the dehydrocondensationreaction starting from galactose in EXAMPLE 2.

[0095]FIG. 10 shows the composition of the oligosacchride containingα-galactosyl obtained in EXAMPLE 2.

[0096]FIG. 11 shows the composition of the oligosacchride containingα-galactosyl obtained in EXAMPLE 4.

[0097]FIG. 12 shows the change with time in the dehydrocondensationreaction starting from galactose in EXAMPLE 5.

[0098]FIG. 13 shows the change in the oligosaccharide employing as astarting material the galactose in EXAMPLE 6.

[0099]FIG. 14 shows the viable cell counts of a Candida microorganism incecum and various colonic positions. “*” indicates a significantdifference observed at a significance level less than 5%.

BEST MODE FOR CARRYING OUT THE INVENTION REFERENCE EXAMPLE 1 Measurementof Activity of α-galactosidase using p-nitrophenyl α-galactoside asSubstrate

[0100] 0.2 ml of 10 mM p-nitrophenyl α-galactoside and 0.2 ml of apredetermined 40 mM buffer (pH in accordance with the optimum pH of theenzyme) were combined with 0.05 ml of an α-galactosidase solution andreacted at 40° C. for 10 minutes. After reaction, 0.5 ml of 0.2 M sodiumcarbonate was added to terminate the reaction, and the liberatedp-nitrophenol was assayed by measuring the absorbance at 400 nm using aspectrophotometer.

[0101] One enzyme activity unit (U) was defined as the level of theenzyme capable of liberating 1 micromole of p-nitrophenol within 1minute under the condition described above.

REFERENCE EXAMPLE 2 Measurement of Activity of α-galactosidase usingMelibiose as Substrate

[0102] 0.2 ml of 10 mM melibiose and 0.2 ml of a predetermined 40 mMbuffer (pH in accordance with the optimum pH of the enzyme) werecombined with 0.05 ml of an α-galactosidase solution and reacted at 40°C. for 10 minutes. Then the mixture was heated at 100° C. for 10 minutesto terminate the reaction, and the resultant glucose level wasquantified using an F-kit manufactured by ROCHE-DIAGNOSTICS(glucose/fructose) or a high pressure liquid chromatography (HPLC).

[0103] One enzyme activity unit (U^(M)) was defined as the level of theenzyme capable of producing 1 micromole of glucose within 1 minute underthe condition described above.

EXAMPLE 1 Preparation of α-galactosidase

[0104] 90 ml of a liquid medium (pH6.0) containing 5% wheat bran wasplaced in a 500 ml Sakaguchi flask, sterilized by a standard method,inoculated with Aspergillus niger strain APC-9319 (FERM BP-7680) andpre-incubated (seed incubation) at 25° C. for 3 days. 500 g of the branwas combined with 400 ml of water, sterilized, inoculated with 10 ml ofthe pre-incubated culture, stirred thoroughly, and subjected to a mainincubation at 25° C. for 4 days.

[0105] After incubation, the bran malt (koji) was pulverized, combinedwith 8 L of water to extract overnight at 4° C., filtered through afilter paper to obtain an extract filtrate. The resultant extractfiltrate was examined for the a-galactosidase activity, which wasrevealed to be 3 U in 1 ml of the extract filtrate. 6 L of the extractfiltrate was concentrated to 1 L using an ultrafiltration membrane (SIPmanufactured by ASAHI KASEI), and combined with ammonium sulfate at aconcentration giving a 70% saturation, whereby effecting a salting out.

[0106] Subsequently, the precipitate was collected by a centrifugation,dissolved in 500 ml of water, concentrated to 100 ml using anultrafiltration membrane, combined further with 500 ml of water,concentrated to 100 ml, this procedure being repeated 3 times, wherebyeffecting a desalting. After desalting followed by a lyophilization, afreeze-dried powder (250 U/g) was obtained.

[0107] The extract filtrate obtained by the above-described method wascombined with ammonium sulfate at a concentration giving a 70%saturation, stirred, and allowed to stand overnight at 4° C. Theprecipitate was collected by a centrifugation, dissolved in 10 mMphosphate buffer (pH6.0), concentrated using an ultrafiltration membrane(SIP manufactured by ASAHI KASEI), combined again with the same buffersolution, and then concentrated. This procedure was repeated threetimes, whereby effecting a desalting.

[0108] Then, an ion exchange column chromatography was conducted using aDEAE-TOYOPEARL 650 M (TOSO) equilibrated with the same buffer solution.Subsequently, a hydrophobic interaction chromatography was conductedusing a butyl-TOYOPERAL 650 M (TOSO) column in 50%-saturated ammoniumsulfate.

[0109] The active fractions were collected, and subjected to a gelfiltration column chromatography using a TOYOPERAL HW-55S (TOSO) columnequilibrated with 50 mM acetate buffer (pH5.5) containing 0.3M sodiumchloride.

[0110] As a result of these column chromatographies, an α-galactosidasewhose proteins are homogeneous was obtained.

[0111] The α-galactosidase obtained as described above is an enzymewhich catalyzes the reaction shown below by which a α-galactoside bondis hydrolyzed to liberate galactose.

Gal1α-OR+H₂O→Gal-OH+R—OH

[0112] wherein Gal1α-OR is a saccharide containing α-galactosyl, Gal-OHis a free galactose, and R—OH is a compound containing hydroxyl such asvarious saccharides, alcohols and phenols.

[0113] Then, this enzyme was employed to measure the optimum pH and thepH stability.

[0114] The enzyme activity in FIG. 1 is measured at 40° C. for 10minutes using various buffer solutions (pH2.0 to 3.5: glycine-HClbuffer, pH3.5 to 6.0: acetate buffer, pH6.0 to 7.5: phosphate buffer).The enzyme activity in FIG. 2 is measured as a residual activity afterallowing the samples to stand at 40° C. for 1 hour using various buffersolutions (pH2.5 to 3.5: glycine-HCl buffer, pH3.5 to 6.0: acetatebuffer, pH6.0 to 8.0: phosphate buffer, pH8.0 to 9.0: Tris-HCl buffer).As a result, the optimum pH of this enzyme was 2.5 to 6.0, and thestability was observed within the range from 3.5 to 8.0 when allowed tostand at 40° C. for 1 hour.

[0115] The optimum temperature and the thermostability of this enzymewere also measured.

[0116] In FIG. 3, the enzyme activity was measured at each temperatureusing an acetate buffer (pH4.5). In FIG. 4, the residual activity aftertreating at each temperature for 15 minutes was measured using theacetate buffer (pH4.5). As a result, the optimum temperature was 60° C.,and the stability was observed at 60° C. or below when allowed to standfor 15 minutes.

[0117] The molecular weight of this enzyme was determined by an HPLC.Using an HPLC column YMC-Pack Diol-200 produced by YMC equilibrated witha 0.1M phosphate buffer solution (pH7.0) containing 0.2M sodium chloridetogether with an MW-Marker (HPLC) produced by Oriental Yeast as astandard protein, the molecular weight was determined. In FIG. 5, thesolid line represents the absorption of the protein at 280 nm, while represents the activity of the α-galactosidase. FIG. 6 shows acalibration curve, the standard proteins employed for which areglutamate dehydrogenase (250,000), lactate dehydrogenase (142,000),enolase (67,000), adenylate kinase (32,000) and cytochrome C (12,000) inthe order of the larger molecular weight.

[0118] The figure in each bracket represents the respective molecularweight. As a result, the molecular weight of this enzyme was 217,000.

[0119] The molecular weight of this enzyme was determined also by anSDS-PAGE. The electrophoresis instrument employed was Phast Systemproduced by Amarsham Pharmacia Biotech. The gel employed wasPhastGelHomogeneous 12.5. The molecular weight markers employed were ofa HMW marker kit (Amarsham Pharmacia Biotech). The standard proteins inthe kit shown in FIG. 7 were myosin (220,000), α-2-macriglobulin(170,000), β-galactosidase (116,000), transferrin (76,000) and glutamatedehydrogenase (53,000) in the order of the larger molecular weight. Thefigure in each bracket represents the respective molecular weight. As aresult, the molecular weight of this enzyme was 117,000.

[0120] The isoelectric point of this enzyme was determined by anisoelectric focusing. The isoelectric point was determined by runningfor 2 days at 500V and 4° C. using *AMPHORITE* produced by AmarshamPharmacia Biotech (pH3.5 to 5.0). In FIG. 8, the solid line representsthe absorption of the protein at 280 nm, while  represents the activityof the α-galactosidase and ⋄ represents the pH. As a result, theisoelectric point of this enzyme was 4.2.

[0121] Among the characteristics described above, the molecular weightof the α-galactosidase produced by this microorganism determined by thegel filtration method (217,000) and that by the SDS-PAGE (117,000) werelarger distinguishably than those of Aspergillus niger-producedα-galactosidase reported previously, i.e., 72,000 and 69,000 (both bySDS-PAGE, Enzyme and microbial Technology, 22:383-390, 1998).

EXAMPLE 2 Production1 of Oligosacchride Containing α-galactosyl

[0122] 100 ml of a buffer solution, pH4.5, containing 60 g of agalactose (WAKO PURE CHEMICAL) and 2,100 U^(M) of the α-galactosidaseobtained in EXAMPLE 1 (galactose concentration: 60% (w/v), enzymeconcentration 35 U^(M)-galactose) was prepared, and allowed to react at50° C. for 30 hours. The change with time during the reaction is shownin FIG. 9.

[0123] The reaction solution was loaded onto an activated charcoalcolumn, from which the galactose was eluted with water and theoligosaccharide was eluted with the gradient of ethyl alcohol from 0 to30%. The eluted oligosaccharide fraction was concentrated into drynessto obtain 24 g of an oligosacchride containing α-galactosyl. Thisoligosaccharide produced only galactose when hydrolyzed with anα-galactosidase or an acid.

[0124] This oligosacchride containing α-galactosyl was subjected to anHPLC analysis (FIG. 10) using a Shim-pack SCR-101 column (SHIMADZU) andrevealed to consist of 11% of oligosaccharides of α-galactotetraose orhigher saccharides, 27% of α-galactotriose and 62% of α-galactobiose.The reaction product was labeled with an aminobenzoic acid ethyl esterand analyzed on an ODS column, which revealed the binding positionisomer composition of the α-galactobiose of α-1,6 bond: α-1,3-bond:α-1,2bond: α-1,4 bond=44:10:5.5:1.0.

COMPARATIVE 1 Production2 of Oligosacchride Containing α-galactosyl

[0125] Using an α-galactosidase produced by Candida guilliermondiistrain H-404 which is known to have the highest dehydrocondensationreaction-catalyzing activity among the α-galactosidases instead of theα-galactosidase obtained in EXAMPLE 1 and the condition similar to thatin EXAMPLE 2, an oligosaccharide was produced.

[0126] As a result, it took 90 hours to observe the reaction plateau (vs30 hours in EXAMPLE 2) in spite of the enzymatic action on melibiose asa substrate similar to that of the α-galactosidase obtained in Example1, and the yield of the oligosacchride containing α-galactosyl was 14 g(vs 24 g in EXAMPLE 2).

[0127] Based on the results described above, the α-galactosidase of theinvention obtained in EXAMPLE 1 was proven to have an extremely highenzymatic activity of the dehydrocondensation reaction using a galactoseas a substrate, and to be able to produce the oligosacchride containingα-galactosyl at a high yield within a short period of time.

EXAMPLE 3 Production3 of Oligosacchride Containing α-galactosyl

[0128] 15 L of a sample containing 9.0 kg of a galactose (WAKO PURECHEMICAL) and 315,000 U^(M)/g of the α-galactosidase obtained in EXAMPLE1 (galactose concentration: 60% (w/v), enzyme concentration 35U^(M)-galactose, pH adjusted at 4.5 with HCl) was prepared, andsubjected to a preliminary reaction for 3 hours at 60° C. in a vacuumcrystallization container (Crystallization instrument, Model DP,manufactured by TSUKISHIMA KIKAI), and then the reaction solution wasconcentrated under reduced pressure at 60° C. to 10 L (galactoseconcentration: 90% (w/v)) and then the concentrate was transferred intoa trough crystallization promoter (TSUKISHIMA KIKAI), to which anα-galactosidase whose enzyme concentration was 35 U^(M)/g-galactose wasadded and the main reaction was effected at 60° C. for 30 hours. Thereaction solution was loaded onto an ion exchange column (ligandexchange column having calcium as a ligand), from which theoligosaccharide and the galactose were eluted with water in this order.The eluted oligosaccharide fraction was concentrated into dryness toobtain 5.1 kg of the oligosacchride containing α-galactosyl.

[0129] By this production method, the substrate can be used at anextremely high concentration (final concentration: 90%), which promotesthe dehydrocondensation reaction of the α-galactosidase extremely, thusenabling an efficient production of an oligosacchride containingα-galactosyl at a high yield within a short period of time, which issuitable especially for an industrial scale production.

[0130] The resultant oligosacchride containing α-galactosyl producedonly galactose when hydrolyzed with an α-galactosidase. Thisoligosacchride containing α-galactosyl was subjected to an HPLC analysisand revealed to consist of 13% of oligosaccharides of α-galactotetraoseor higher saccharides, 31% of α-galactotriose and 56% of α-galactobiose.The binding position of the α-galactosyl group at the reducing terminalwas mainly of α-1,6 bond.

EXAMPLE 4 Production4 of Oligosacchride Containing α-galactosyl

[0131] 100 g of a lactose (WAKO PURE CHEMICAL) was hydrolyzed with acommercially available β-galactosidase (LACTOZYME produced byNOVONORDISC BIOINDUSTRIES) to obtain a mixture of equal amounts of agalactose and a glucose. 100 ml of an acetate buffer, pH4.5, containing85 g of the equal amount mixture of the galactose and the glucoseobtained above and 1,500 U^(M) of the α-galactosidase obtained inEXAMPLE 1 (saccharide concentration: 85% (w/v), enzyme concentration 35U^(M)/g-galactose) was prepared, and allowed to react at 50° C. for 87hours.

[0132] From the reaction solution, 27 g of an oligosacchride containingα-galactosyl was obtained by a column chromatography on an activatedcharcoal. This oligosacchride containing α-galactosyl produced only thegalactose and the glucose, when hydrolyzed with an acid orα-galactosidase, with the ratio between the galactose and the glucosebeing about 7:3. This oligosacchride containing α-galactosyl subjectedto an HPLC analysis (FIG. 11) and revealed to consist of 8.0% oftrisaccharides or higher oligosaccharides, 48% of α-galactosyl glucoseand 44% of α-galactobiose.

COMPARATIVE 2 Production5 of Oligosacchride Containing α-galactosyl

[0133] Using an α-galactosidase produced by Candida guilliermondiistrain H-404 instead of the α-galactosidase obtained in EXAMPLE 1 andthe condition similar to that in EXAMPLE 4, an oligosaccharide wasproduced. As a result, the yield of the oligosacchride containingα-galactosyl was 16 g (vs 27 g in EXAMPLE 4).

[0134] Based on the results described above, the α-galactosidase of theinvention obtained in EXAMPLE 1 was proven to have an extremely highenzymatic activity of the dehydrocondensation reaction even when using amixture of the galactose and the glucose as a substrate, and to be ableto produce the oligosacchride containing α-galactosyl at a high yield.

EXAMPLE 5 Production6 of Oligosacchride Containing α-galactosyl

[0135] 100 ml of an acetate buffer solution, pH4.5, containing 60 g of agalactose (WAKO PURE CHEMICAL) and 2,100 U^(M) of an α-galactosidaseS-DS derived from Aspergillus niger (AMANO ENZYME) (galactoseconcentration: 60% (w/v), enzyme concentration 35 U^(M)/g-galactose) wasprepared, and allowed to react at 50° C. for 30 hours. The change duringthe reaction is shown in FIG. 12.

[0136] The reaction solution was loaded onto an activated charcoalcolumn, from which the galactose was eluted with water and theoligosaccharide was eluted with the gradient of ethyl alcohol from 0 to30%. The eluted oligosaccharide fraction was concentrated into drynessto obtain 21 g of an oligosacchride containing α-galactosyl. Thisoligosaccharide produced only galactose when hydrolyzed with anα-galactosidase or an acid. This oligosacchride containing α-galactosylwas subjected to an HPLC analysis and revealed to consist of 9.0% ofoligosaccharides of α-galactotetraose or higher saccharides, 25% ofα-galactotriose and 66% of α-galactobiose.

EXAMPLE 6 Production7,8,9 of Oligosacchride Containing α-galactosyl

[0137] 100 ml of an acetate buffer, pH4.5, containing 20 g of agalactose (WAKO PURE CHEMICAL) and each 400 U^(M) of the α-galactosidaseobtained in EXAMPLE 1 or the α-galactosidase purified by a known methodfrom Penicillium citrinum-derived protease B “AMANO” or nuclease “AMANO”(galactose concentration: 20% (w/v), enzyme concentration 20U^(M)/g-galactose) was prepared, and allowed to react at 40° C. Thechange with time during the reaction is shown in FIG. 13.

[0138] While the results indicated no difference in the trisaccharideratio between enzymes, the disaccharide ratios after reaction for 100hours given by the α-galactosidase obtained in EXAMPLE 1, nuclease“Amano” and protease B “AMANO” were 10.0%, 11.3% and 11.1%,respectively, showing the difference. As evident from these results,there was a difference in the compositions of he galactoses havingdifferent binding numbers among the various origins of theα-galactosidase even when the reaction was conducted under the identicalcondition, and thus the α-galactosidase derived from Penicilliumcitrinum is preferable for the purpose of obtaining an oligosaccharidewhose ratio of the disaccharides is high.

EXAMPLE 7 Anti-Candida Effect of Oligosacchride Containing α-galactosyl

[0139] A Candida microorganism was given forcibly via an oraladministration to a mouse on diet with the oligosacchride containingα-galactosyl obtained in EXAMPLE 3 to test the persistence of theCandida microorganism in an intestinal tract, whereby assessing theanti-candida effect of the oligosacchride containing α-galactosyl.

[0140] (Methods) A test mouse was a 4-week old male mouse of Crj:CD-1(ICR) line (NIPPON CHARLES RIVER). A preliminary housing was provided tothe animal for 1 week while being allowed to access a basal diet and adistilled water ad libitum. The basal diet was a solid chow for mice(MF: ORIENTAL YEAST). After the preliminary housing, the basal diet andthe distilled water were given ad libitum continuously, and a controlgroup fed with a sucrose, a positive control group fed with a raffinose,and a treatment group fed with an oligosacchride containing α-galactosylwere provided (5 animals per group) to conduct a forcible oraladministration at 100 mg/animal via an oral gavage for two weeks on aonce-a-day basis. One week after initiation of the treatment, theanimals in each group received 0.2 ml of a suspension of a Candidamicroorganism (Candida albicans IFM40009) prepared at the density of5.0×10⁹ cells/ml, 1 week after which the cecum and the colon of eachmouse were isolated and examined for the viable Candida cell count ofeach organ.

[0141] The viable cell of the Candida microorganism was counted byapplying a Candida microorganism suspension diluted appropriately with aphysiological saline onto a Candida GS (EIKEN KAGAKU) plate medium andincubating at 37° C. for 42 to 48 hours followed by counting thecolonies formed. The statistical processing was conducted using aWilcoxson's rank sum test.

[0142] (Results) The viable cell counts of the cecum and the each partof the colon 1 week after the forcible administration of the Candidamicroorganism suspension are shown in FIG. 14. While the viable cellcount of the cecum 1 week after the forcible administration of theCandida microorganism suspension was not changed in the control group orthe positive control group but was lower in the treatment group, that ofthe colon was lower in both of the positive control group and thetreatment group, with a significantly lower count being observed in thetreatment group especially when compared with the control group. In thefigure, α-GOS is an oligosacchride containing α-galactosyl.

[0143] Based on the results, the oligosacchride containing α-galactosylin the treatment group exhibited a potent inhibitory effect on thesettlement of the Candida microorganism in the intestinal tract, andrevealed to have a more excellent anti-candida effect than raffinose.

INDUSTRIAL APPLICABILITY

[0144] Since a method of elevating the yield of an oligosacchridecontaining α-galactosyl according to the present invention is capable ofproducing any of various oligosaccharide efficiently at a high yieldwithin a short period of time and is also capable of producing on anindustrial scale, it enables a stable and less expensive supply ofoligosaccharide.

[0145] Also since an anti-candida composition of the invention has anexcellent anti-candida effect and can be supplied stably, it can providea less expensive pharmaceutical or functional food material whichenables a safe amelioration or prevention of a Candidamicroorganism-induced mycosis or allergic disease such as an atopicdermatitis.

What is claimed is:
 1. A method of elevating the yield of anoligosacchride containing α-galactosyl comprising treating galactose ora galactose-containing material with an α-galactosidase derived from amicroorganism of Aspergillus, Penicillium, Trichoderma, Saccharomyces orBacillus.
 2. A method of elevating the yield of an oligosacchridecontaining α-galactosyl according to claim 1 wherein the α-galactosidaseis derived from a microorganism of Aspergillus.
 3. A method of elevatingthe yield of an oligosacchride containing α-galactosyl according toclaim 2 wherein the α-galactosidase is derived from Aspergillus niger.4. A method of elevating the yield of an oligosacchride containingα-galactosyl according to claim 3 wherein the α-galactosidase has thefollowing physicochemical characteristics: [1] Effect It catalyzes areaction for hydrolyzing an α-galactoside bond to liberate D-galactose:Gal1α-OR+H₂O→Gal-OH+R—OH wherein Gal1α-OR is a saccharide containingα-galactosyl, Gal-OH is a free galactose, and R—OH is a compoundcontaining hydroxyl such as various saccharides, alcohols and phenols);[2] Substrate specificity It acts on a saccharide having an α-galactosylgroup at its non-reducing terminal such as melibiose, raffinose andstachyose as well as p-nitrophenyl α-galactoside, with the relative rateat which melibiose is decomposed being about 9 based on thedecomposition rate using p-nitrophenyl α-galactoside as a substratebeing regarded as 100; [3] Optimum pH and pH stability Its optimum pH is2.5 to 6.0, and it is stable within the range from pH 3.5 to 8.0 whenallowed to stand for 1 hour at 40° C.; [4] Optimum temperature and heatstability Its optimum temperature at pH 4.5 (acetate buffer) is 60° C.,and it is stable at a temperature not higher than 60° C. when allowed tostand at pH 4.5 (acetate buffer) for 15 minutes; [5] Molecular weightand isoelectric point Its molecular weight measured by a gel filtrationmethod using a YMC-Pack Diol-200 column (YMC) is 217,000 and itsmolecular weight measured by an SDS-PAGE is 117,000, while itsisoelectric point measured by an isoelectric focusing is 4.2.
 5. Amethod of elevating the yield of an oligosacchride containingα-galactosyl according to claim 4 wherein the α-galactosidase having thephysicochemical characteristics described above is derived fromAspergillus niger strain APC-9319 (FERM BP-7680).
 6. A method ofelevating the yield of an oligosacchride containing α-galactosylcomprising treating the galactose or a galactose-containing materialwith an α-galactosidase to effect a dehydrocondensation reaction as apreliminary reaction followed by concentrating the reaction solutionunder reduced pressure prior to the dehydrocondensation reaction as amain reaction.
 7. A method of elevating the yield of an oligosacchridecontaining α-galactosyl according to claim 6 comprising treating with anα-galactosidase derived from Aspergillus niger strain APC-9319 (FERMBP-7680) having the physicochemical characteristics described above. 8.An α-galactosidase produced by Aspergillus niger strain APC-9319 (FERMBP-7680) having the physicochemical characteristics described above. 9.A method of producing an α-galactosidase according to claim 8 comprisingincubating Aspergillus niger strain APC-9319 (FERM BP-7680) in a culturemedium and isolating the α-galactosidase from the culture. 10.Aspergillus niger strain APC-9319 (FERM BP-7680) producing anα-galactosidase according to claim
 8. 11. An anti-candida compositioncomprising as an active ingredient an oligosacchride containingα-galactosyl other than raffinose which is synthesized by treatinggalactose or a galactose-containing material with an α-galactosidase toeffect a dehydrocondensation reaction.
 12. An anti-candida compositioncomprising as an active ingredient an oligosacchride containingα-galactosyl other than raffinose which is synthesized by a method ofelevating the yield according to any of claims 1 to
 7. 13. Ananti-candida composition according to claim 11 or 12 wherein theoligosacchride containing α-galactosyl is α-(Gal)n wherein n is usuallyan integer of 2 to 10, and Gal represents galactose).